Protein modification with ubiquitin plays key regulatory roles in a wide variety of biological phenomena including tumorigenesis. Several oncoproteins and anti-oncoproteins display ubiquitin ligase activity and identification of their substrates is one of the central themes in cancer research. This application is directed toward understanding the biological roles of a tumor-suppressing ubiquitin ligase, VHL. VHL is currently best understood as a negative regulator of hypoxia inducible factor (HIF). In the presence of oxygen and iron, specific proline residues in HIF are hydroxylated and these hydroxylated prolines are recognized by VHL, which results in ubiquitination and degradation of HIF. VHL mutation is associated with hereditary and sporadic renal cell carcinomas, hemangioblastomas, and pheochromocytomas. Several lines of evidence suggest that, in addition to HIF, there are unidentified substrates for VHL which play critical roles in tumor suppression. A major barrier to understanding the VHL functions (as well as the functions of other ubiquitin ligases) has been the difficulty in pinpointing its ubiquitination substrates. Employing a novel quantitative proteomics technology, ICAT (isotope-coded affinity tag), we developed an approach to systematically identify the substrates of ubiquitin ligases and using this method, we identified a novel substrate for VHL, which carries the character of a key mediator of VHL functions. Therefore the aims of this application are to analyze the mechanism and biological consequence of ubiquitination of this substrate and to conduct further proteomic screens to identify additional substrates for VHL. These studies should provide a better understanding of the biological functions of VHL and advance the nascent field of proteomics-based biology.